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1.
J Vis Exp ; (129)2017 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-29155743

RESUMO

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that <40 µm alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.


Assuntos
Imunoensaio/métodos , Análise Serial de Proteínas/métodos , Proteínas/metabolismo , Reações Cruzadas , Humanos , Proteínas/análise
2.
Traffic ; 17(4): 416-32, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26756199

RESUMO

Calnuc is a ubiquitous Ca(2+)-binding protein present on the trans-Golgi network (TGN) and endosomes. However, the precise role of Calnuc in these organelles is poorly characterized. We previously highlighted the role of Calnuc in the transport of LRP9, a new member of a low-density lipoprotein (LDL) receptor subfamily that cycles between the TGN and endosomes. The objective of this study was to explore the role of Calnuc in the endocytic sorting of mannose-6-phosphate receptor (MPR) and Sortilin, two well-characterized lysosomal receptors that transit between the TGN and endosomes. Using biochemical and microscopy assays, we showed that Calnuc depletion [by small interfering RNA (siRNA)] causes the misdelivery to and degradation in lysosomes of cationic-independent mannose-6-phosphate receptor (CI-MPR) and Sortilin due to a defect in the endosomal recruitment of retromers, which are key components of the endosome-to-Golgi retrieval machinery. Indeed, we demonstrated that Calnuc depletion impairs the activation and membrane association of Rab7, a small G protein required for the endosomal recruitment of retromers. Overall, our data indicate a novel role for Calnuc in the endosome-to-TGN retrograde transport of lysosomal receptors through the regulation of Rab7 activity and the recruitment of retromers to endosomes.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endossomos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptor IGF Tipo 2/metabolismo , Animais , Células COS , Proteínas de Ligação ao Cálcio/genética , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Proteínas do Tecido Nervoso/genética , Nucleobindinas , Transporte Proteico , Rede trans-Golgi/metabolismo
3.
Bioconjug Chem ; 26(3): 405-11, 2015 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-25654426

RESUMO

Arginine-rich cell penetrating peptides are short cationic peptides able to cross biological membranes despite their peptidic character. In order to optimize their penetration properties and further elucidate their mechanisms of cellular entry, these peptides have been intensively studied for the last two decades. Although several parameters are simultaneously involved in the internalization mechanism, recent studies suggest that structural modifications influence cellular internalization. Particularly, backbone rigidification, including macrocyclization, was found to enhance proteolytic stability and cellular uptake. In the present work, we describe the synthesis of macrocyclic arginine-rich cell penetrating peptides and study their cellular uptake properties using a combination of flow cytometry and confocal microscopy. By varying ring size, site of cyclization, and stereochemistry of the arginine residues, we studied their structure-uptake relationship and showed that the mode and site of cyclization as well as the stereochemistry influence cellular uptake. This study led to the identification of a hepta-arginine macrocycle as efficient as its linear nona-arginine congener to enter cells.


Assuntos
Membrana Celular/metabolismo , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Células HeLa , Humanos , Relação Estrutura-Atividade
4.
Hum Mutat ; 34(12): 1688-97, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24038957

RESUMO

One late infantile variant of the neurodegenerative disease neuronal ceroid lipofuscinosis (NCL) is caused by a mutation in the CLN5 gene. CLN5 encodes a lysosomal glycoprotein whose structure and function have not yet been clearly defined. In the present study, we used epitope-tagged CLN5 to determine the topology and solubility of the CLN5 protein. Our results indicated that CLN5 is synthesized as a type II transmembrane (TM) glycoprotein with a cytoplasmic N-terminus, one TM segment, and a large luminal C-terminal domain containing an amphipathic helix (AH). The cytoplasmic and TM domains were rapidly removed following signal-peptide cleavage, and the resulting mature CLN5 was tightly associated with the lumen of the membrane through the AH. CLN5 pathological mutants deprived of AH lose their membrane association, are retained in the endoplasmic reticulum, and are rapidly degraded by the proteasomal machinery. We experimentally define the topology of CLN5 and demonstrate the existence of an AH that anchors the protein to the membrane. Our work sheds light on the basic properties of CLN5 required to better understand its biological functions and involvement in NCL pathogenesis.


Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/genética , Sequência de Aminoácidos , Linhagem Celular , Expressão Gênica , Humanos , Proteínas de Membrana Lisossomal , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência
5.
Traffic ; 10(8): 1098-114, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19497050

RESUMO

Calnuc is an ubiquitous Ca(++)-binding protein found in the cytoplasm where it binds different Galpha subunits, in the Golgi lumen where it constitutes a major Ca(++) storage pool, and outside the cell. We identified LDLR-related protein 9 (LRP9) as the first transmembrane protein shown to interact directly with Calnuc. LRP9 is a member of a new subfamily of the LDLR superfamily that cycles between the trans-Golgi network (TGN) and endosomes through a mechanism dependent on clathrin adaptor GGA proteins. The aim of the present study was to characterize the interaction between Calnuc and LRP9. Various biochemical assays showed that the N-terminus of Calnuc interacts with an arginine-rich region in the cytosolic tail of LRP9. Confocal microscopy showed that Calnuc colocalizes with LRP9 at the surface of the TGN and early endosomes. Depletion of Calnuc by small interfering RNA (siRNA) missorted LRP9 in the late endosome/lysosome compartments and enhanced its lysosomal degradation. This phenotype was rescued by the expression of siRNA-resistant wild-type Calnuc as well as cytoplasmic Calnuc, indicating that the cytoplasmic pool of Calnuc is involved in LRP9 endosomal sorting to prevent the delivery of LRP9 to lysosomes. This is the first report showing that Calnuc plays a role in receptor trafficking.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endossomos/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transporte Proteico/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular , Proteínas de Ligação a DNA/genética , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Proteínas Relacionadas a Receptor de LDL/genética , Lisossomos/metabolismo , Proteínas de Membrana Transportadoras/genética , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso , Nucleobindinas , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Técnicas do Sistema de Duplo-Híbrido , Rede trans-Golgi/metabolismo
6.
Histochem Cell Biol ; 130(2): 315-27, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18461348

RESUMO

LDL receptor-related protein 9 (LRP9) is a distant member of the low-density lipoprotein receptor (LDLR) superfamily. To date, there are no reports on the cellular distribution of LRP9 or the signals responsible for its localization. Here, we investigated the intracellular localization and trafficking of LRP9. Using confocal microscopy, we demonstrated that LRP9 was not present at the plasma membrane but co-localized with various markers of the trans-Golgi network (TGN) and endosomes. This co-localization was dependent on the presence of two acidic cluster/dileucine (DXXLL) motifs in the cytoplasmic tail of LRP9, which interact with GGA proteins, clathrin adaptors involved in transport between the TGN and endosomes. LRP9 is the first example of a transmembrane protein with an internal GGA-binding sequence in addition to the usual C-terminal motif. An inactivating mutation (LL --> AA) in both DXXLL motifs, which completely inhibited the interaction of LRP9 with GGA proteins, led to an intracellular redistribution of LRP9 from the TGN to early endosomes and the cell surface, indicating that the two DXXLL motifs are essential sorting determinants of LRP9. In conclusion, our results suggest that LRP9 cycles between the TGN, endosomes and the plasma membrane through a GGA dependent-trafficking mechanism.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Endossomos/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Mutantes/metabolismo , Rede trans-Golgi/metabolismo , Motivos de Aminoácidos , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Citoplasma/metabolismo , Proteínas Relacionadas a Receptor de LDL/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteínas de Membrana Transportadoras/genética , Camundongos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Transporte Proteico , Transfecção
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